To develop a model system in which we could induce GCRs, we used the rare restriction enzyme I-SceI, whose 18 bp recognition sequence is not normally present in the human or mouse genome, to produce a single DNA DSB within a mammalian cell, based on the hypothesis that improper repair of these breaks could lead to GCRs. This enzyme has been used in a series of elegant studies to produce specific, non-random GCRs mediated by homologous recombination in mammalian cells. We generated a construct that expressed the Herpes simplex virus type I thymidine kinase (TK) gene under the control of the constitutive EF1a promoter, with the recognition sequence for the I-SceI restriction enzyme placed between the EF1a promoter and the TK gene. This pEF1aTK vector was introduced into the U937 cell line, and verified that expression of the TK gene conferred sensitivity to ganciclovir (GCV). We then carried out a series of experiments that utilized the negative selection provided by the expression of TK. Cells were transfected with an I-SceI expression vector and selected with GCV (to select for cells that had lost TK expression). The vast majority of the clones had small, interstitial deletions, and no clones showed GCR. However, 8 clones showed small DNA segments (50-1500 bp) that were derived from distant regions of the genome inserted at the DNA DSB site; further studies recovered over 100 clones with insertions. Most of these insertions were derived from transcribed regions of the genome, leading to the hypothesis that these DNA DSB may have been repaired by patches generated from reverse transcribed RNA. To determine whether this form of DNA repair was restricted to experimentally induced DNA DSB, we analyzed whole genome sequence data from myeloma cell lines, and identified 23 instances of insertions derived from distant genomic regions (which we have termed template sequence insertions, or TSIs). Surprisingly, identical TSIs were seen in normal individuals, indicating that most TSIs were polymorphic in the human germline. Approximately half of the TSIs showed hallmarks of LINE-1 retrotransposon-mediated insertions, such as a 5'-TTTT/A-3' integration site, a target site duplication (TSD), a polyA tract at the insertion site, and a polyadenylation signal. However, the inserted sequence was not a LINE-1 nor a SINE, but instead was a transcribed, genic region that mapped to a distant genomic region. The presence of these L1 ORF1 hallmarks strongly suggests that these TSIs were caused by the LINE-1 integrase and reverse-transcriptase acting on nuclear pre-mRNA. A second class of TSIs showed no preference for a 5'-TTTT/A-3' integration site, no TSD, no poly A tract, but instead showed several bp of microhomology at the insertion junction. This second class is remarkably similar to insertions at I-SceI induced DNA DSB, and we predict that these insertions were also caused by reverse transcription of pre-mRNA creating a patch for a spontaneous DNA DSB that occurred in a germ cell. We suspected that these TSIs could be templated by RNA intermediates, as the genomic regions were not lost from their wild-type chromosomal location. To address this hypothesis, we repeated the I-SceI experiment several times, modified by co-transfection of mouse RNA with the I-SceI expression vector. Approximately 10% of the TSIs recovered from these experiments contained mouse sequences, indicating that they were derived from mouse RNA. To gain a broader insight into the nature of these TSIPs, we studied a publicly available database of whole genome sequence from 52 individuals, and identified approximately 200 TSIPs. Not surprisingly, specific TSIPs tracked with known patterns of human migration. Interestingly, we identified over 20 TSIPs that were derived from mitochondrial DNA, demonstrating that mitochondrial sequences can insert into nuclear sequences, and a subset of TSIPs could be linked to human disease, due to insertions into critical coding regions. These results were published in 2014 and 2015. It may not be surprising that we were unable to generate GCRs by inducing a single DNA DSB, as other investigators have concluded that two induced breaks are required to produce a chromosomal translocation, and that the frequency of chromosomal translocations induced by a single DNA DSB in mouse embryonic stem (ES) cells is extraordinarily rare. To determine whether GCR cold be produced in cells that were defective in DNA DSB repair, we repeated the I-SceI cleavage experiments using cells that were deficient for H2AX, a histone variant that becomes gamma-phosphorylated in response to DNA DSB, and coats the region of the DNA DSB. We inserted the EF1aTK vector into H2AX knockout (KO) cells, and then transfected an I-SceI expression vector. Although the majority of clones resulting from this experiment were vector capture events, in which the DNA DSB had become repaired by transfected plasmid DNA, approximately 15% of the clones had undergone a GCR (balanced translocation or megabase inversion), demonstrating that a GCR can be caused by a single, induced DNA DSB. A manuscript describing these findings will be submitted in the near future.